Conventional methods of gene therapy frequently use plasmid DNA as vectors for introducing desired DNA segments into target cells. In such applications, however, plasmids frequently have certain disadvantages—compared to viral vectors—, of lower transfection efficiency; viral vectors are therefore preferably used.
Until now, about 25% of all gene therapy protocols that have been used in clinical trials are based directly on plasmid DNA vectors (Edelstein et al, J. Gene Med. 2007; 9: 833-42). It was initially expected that the market share of plasmid DNA-based vector vaccines would increase to about 60% (Jain, “Vectors for gene therapy: Current status and future prospects”, PJB Publications Ltd, London, 1996). This includes plasmid DNA used for the production of viral vectors, e.g. by transient transfection of producer cells for adeno-associated (AAV) vectors, lentiviral (LV) vectors, retroviral (RV) vectors or adenoviral (Ad) vectors. The use of at least one plasmid for the production of viral vectors and viruses, for example, has already been described for AAV (WO 03/016521 A2), but also the distribution in different (at least two, but possibly more) plasmids of the above-mentioned viruses or viral vectors has been described. In such cases, the co-transfection of cells with plasmids is performed.
The AAV packaging/helper system based on two plasmids from the laboratory of Jurgen Kleinschmidt (Grimm et al, Hum Gene Therapy 1998; 9: 2745-60) was initially developed for serotypes 1-6. Mutants with e.g. a heparin binding site deficiency (pDG(R484E/r585E), Kern et al, J. Virol. 2003; 77: 11072-81) and other, including synthetic, serotypes for co-transfection are available with only the transfer plasmid (containing the ITRs) on the one hand, and with the packaging/helper plasmid (both functions on another plasmid with a size of about 20 kbp), on the other hand. Other versions of such systems have been published (Lock et al., Hum. Gene Ther. 21, 1273-1285), and two international reference standards have been applied to ensure adequate clinical preparation of the AAV by using the pDG-plasmid system (Moullier and Snyder, Mol Ther 2008, 16: 1185-1188). In such cases, the optimization of transfection is relatively simple, since only the correct ratio of the amounts of both plasmids must be determined when work is resumed using a new batch of the plasmids. This is a far more difficult task when three (or more) plasmids must be triple-transfected and the individual relative amounts of each must therefore be newly optimized when a fresh batch is used. An overview is provided by Ayuso et al. (Curr Gene Ther 2010, 10: 423-436).
In wild-type viruses the cotransfection transfer plasmids for AAV production contain the sequences encoding for the replication and envelope proteins (rep and cap) between the ITR sequences. This area including the rep and cap genes was relocated on other plasmids/the other plasmid cotransfection as part of the development of systems for the production of AAV vectors to create on the transfer plasmid a location for the sequences of interest, which are subsequently supposed to find a place in the viral particle. Expression of these sequences will initially be delayed in the conventional viral AAV vectors after their use for infection of a target cell (such as in uses for gene therapy), since the synthesis of the second strand of DNA (the DNA contained in the viral particles is single-stranded) can only be manufactured with the help of the cellular replication system; thus, enabling only the formation of a transcriptionally competent duplex. The development of so-called self-complementary AAV vectors (Heilbron and Weger, Handb Exp Pharmacol 2010, 197: 143-70) solves this problem through the use of double-stranded “genomes” in the vectors (ITR-flanked sequences of interest). It has been found that these are available as a double strand in the target cell immediately following vector infection. Such double-stranded viral sequences were obtained by deletion of the “terminal resolution site” in an ITR, and during replication the rep proteins were no longer able to cut this DNA for incorporation into the viral particles. Therefore, the replication proceeded across this modified ITR and resulted—using the newly synthesized strand as a template—in a complementary strand. The resulting DNA strand in the forward portion of the sense strand consisted of the sequence of interest and—not interrupted by the unresolved modified ITR—of the antisense strand of the sequence of interest. The generated viral vectors are superior to previous non-self-complementary vectors with respect to their transgene expression (D M McCarty et al, Gene Ther 2003, 10 (26): 2112 to 2118; Z. Wang et al, Gene Ther 2003, 10 (26): 2105-2111).
After Chadeuf et al. (Mol Ther 2005; 12: 744-53) demonstrated that the structural elements of plasmid vectors for the production of AAV particles, namely elements of the transfer plasmid carrying antibiotic resistance genes, were detectable in virus preparations, various regulatory authorities strongly demanded avoiding such sequences in AAV preparations. This recurring problem is also referred to as “retro packaging” and means that individual sequence portions of those plasmids carrying the signal structures for partial packing in viruses or viral vectors (so-called “transfer plasmids”, sometimes referred to as “vector plasmids”) are incorrectly packed into the viruses or viral vectors. A transfer plasmid contains its regulatory elements (bacterial origin of replication and selection marker) and the sequences of interest to be transferred (e.g. a gene). These sequences of interest are flanked by signal sequences, according to the prior art, e.g. so-called ITRs, or inverted terminal repeats, in AAV; or LTRs, which are long terminal repeats, in LV. However, since an (intact) plasmid is constructed as a circle, the framing of a sequence of interest to the exclusion of origin of replication and/or selection marker means that excluded elements, or at least one thereof (origin of replication and/or selection marker) on the reverse side of the plasmid also are flanked by these signal sequences. Thus, the encoded sequences (also shown for AAV, see Chadeuf et al. 2005) can also be packaged in the viral capsids, albeit at a slower rate, and lead to nonfunctioning or even dangerous viral vectors. These are detectable in preparations of viruses or viral vectors and can also additionally lead to a pharmaceutical threat of a mixture of functional and non-functional viruses or viral vectors—accordingly, with reduced efficiency.
The above situation has led to the development of a minicircle system, as disclosed herein, that avoids the aforementioned problems in the future.
Recently, so-called minicircles (MC), small circular DNA molecules containing a desired expression cassette and a few undesirable prokaryotic sequences, have been used to transfect cells. One method for the production of minicircles is described in WO 96/26270. It was further demonstrated that minicircles offer, apart from improved biosafety due to their small size, improved gene transfer characteristics (A. M. Darquet et al., Gene Ther. 1997, 4: 1341-1349; A. M. Darquet et al., Gene Ther. 1999, 6: 209-218).
Bigger et al (J. Biol Chem 2001, 276: 23018-23027) describe the preparation of minicircles by means of the introduction of plasmids with loxP sites in bacteria, which can express the Cre recombinase. The plasmid further comprises a eukaryotic expression cassette and a marker sequence. After induction of Cre the plasmid is cleaved into miniplasmid and minicircle, wherein the minicircle contains only the expression cassette. In addition, the loxP sites are mutated, so that the reversibility of recombination is reduced.
Other publications also describe the production of minicircles using alternative recombination systems, e.g. Kreiss et al. (Appl Microbiol Biotechnol 1998, 49: 560-567) using λ integrase and Chen et al. (Mol Ther 2003, 8: 495-500) using ΦC31 integrase. Therefore, minicircles are established as alternative vectors used for transfecting eukaryotic cells.